Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle

Blastema formation is a crucial process that provides a cellular source for regenerating tissues and organs. While bilaterians have diversified blastema formation methods, its mechanisms in non-bilaterians remain poorly understood. Cnidarian jellyfish, or medusae, represent early-branching metazoans that exhibit complex morphology and possess defined appendage structures highlighted by tentacles with stinging cells (nematocytes). Here, we investigate the mechanisms of tentacle regeneration, using the hydrozoan jellyfish Cladonema pacificum. We show that proliferative cells accumulate at the tentacle amputation site and form a blastema composed of cells with stem cell morphology. Nucleoside pulse-chase experiments indicate that most repair-specific proliferative cells (RSPCs) in the blastema are distinct from resident stem cells. We further demonstrate that resident stem cells control nematogenesis and tentacle elongation during both homeostasis and regeneration as homeostatic stem cells, while RSPCs preferentially differentiate into epithelial cells in the newly formed tentacle, analogous to lineage-restricted stem/progenitor cells observed in salamander limbs. Taken together, our findings propose a regeneration mechanism that utilizes both resident homeostatic stem cells (RHSCs) and RSPCs, which in conjunction efficiently enable functional appendage regeneration, and provide novel insight into the diversification of blastema formation across animal evolution.

incubation time from 1 hour to 24 hours and found that 13.6% of the BrdU + cells were EdU + resident stem cells in the 1 hour EdU incubation, while 26.9% of the BrdU + cells were EdU + cells in the 24 hour EdU incubation (Revised Fig. 4Cii, 4D, 5Bii, 5Biv, and 5C).This result shows that even under 24-hour conditions in which most resident stem cells must be labeled, more than 70% of the proliferative cells are distinct from resident stem cells.These results together strongly suggest that migration of resident stem cells into the bulb is not sufficient to form blastema.We have added the following sentences to the text in the revised manuscript.
(Revised Manuscript; Page 18, Line 622-624) "Importantly, only 13.6% of BrdU + RSPCs were EdU + in blastema, supporting the notion that the migration of RHSCs may not be sufficient to form blastema (Fig 4Bi ,   4Cii, and 4D)." (Revised Manuscript; Page 20, Line 681-687) "After EdU labeling for 24 h, followed by amputation, most EdU + cells remained in the bulb, and 26.9% of the BrdU + RSPCs were EdU + in the blastema, indicating that the majority of RSPCs are different lineage than RHSCs (Fig 5B ii, 5Biv, and 5C).This result is consistent with the results of the chasing experiment using short duration EdU labeling (Fig 4A -4D), further supporting the conclusion that the migration of RHSCs in the bulb is not sufficient to form blastema." Comment 1.1.3:Not all tentacle bulb S-phase cells are stem cells; many of them are transit amplifying progeny (e.g., nematoblasts) that are not expected to contribute to the cycling cells in the blastema (i.e., incorporate BrdU) 24 hpa because they would exit the cell cycle within 24 hours after EdU labeling.This would reduce the number of expected double labeled cells under the migration hypothesis, making the observed 12.6% inconsistent with the authors' dedifferentiation model.

Response 1.1.3
It is true that the transit amplifying progeny including nematoblasts among EdU + cells would not be co-labeled with BrdU in blastema once they exit the cell cycle.However, nematoblasts must be considered if they migrate to the blastema region.As we have stated in Response 1.1.2,migration of resident stem cells is not a major determinant of blastema formation.Furthermore, in the revised manuscript, we have performed detailed cell tracking in the bulb, including resident stem cells by DiI injection, and shown that the labeled cells do not migrate to the blastema from the time of amputation to 24 hpa (Revised Fig. S6A-S6G).Based on these results, we consider that the transit amplifying progeny like nematoblasts generally do not participate in blastema formation in addition to resident stem cells.As pointed out, repair-specific proliferative cells can arise not only from the dedifferentiation of neighboring differentiated cells but also from the cell cycle re-entry of quiescent/slow cycling cells.In fact, considering this possibility, we clearly described these two possibilities for the origin of repair-specific proliferative cells in the Discussion of our manuscript.However, as we mentioned in the Response 1.1.1,we cannot discriminate these two possibilities because the current available tools do not allow us to track a specific cell lineage in the Cladonema medusa and thus left as they are for future studies.
Comment 1.1.5:Figure 2 i' and I'' show that S-phase in the blastema at 24 hpa comes at the expense of cycling cells in the tentacle bulb.This observation is consistent with a scenario in which many cycling tentacle bulb i-cells had migrated to the blastema.If dedifferentiation were the case, one would not expect tentacle bulb i-cells to reduce their proliferation rates.

Response 1.1.5
In the original Figure 2A(ii) that showed the regenerating tentacle at 24 hpa, fewer EdU + cells were distributed in the bulb, and most EdU + cells accumulated in the amputated region, so it seems that the reviewer concluded that S-phase cells in the blastema comes at the expense of cycling cells in the tentacle bulb.However, this is likely due to the fact that we showed the EdU images of 24-72 hpa (Figure 2Aii-iv in the original manuscript), which were the results of 1 h incubation with EdU 20 µM, not the results of 1 h incubation with EdU 150 µM, which were used in other experiments.Another issue is that the signal of EdU in the tentacle bulb, which corresponds to resident stem cells, was not so high compared to blastema, so the brightness of these EdU signal was adjusted by ImageJ/Fiji for better visibility (Revised Fig. 2Ai).On the other hand, the signal of EdU was quite strong in blastema at 24 hpa, and we tried to avoid saturation of the signal intensity at blastema, which caused the signal in the bulb to become darker.This may have given the impression that EdU + cells in the bulb were moving to the injury site.
In the revised manuscript, we have replaced these images with the ones obtained by 1h incubation with EdU 150 µM (Revised Fig. 2A, 2B), which are consistent with other EdU pulse labeling experiments (Revised Fig. 2D, 2G-2I, 3E, 4A-4C, 4I, 5B, 5F, 6B, 6E, S2C, S4C-S4D, S5A-S5C, S6B, S6E, S8A, S10A-S10D, and S11C).As a result, EdU + cells in the bulb were clearly detected at 24 hpa, although the signal of these EdU + cells was weaker than that of blastema (Revised Fig. 2Aii).We have also quantified the relative intensity of EdU signal (Fig. R1) and found a strong signal as the evidence of highly proliferative population.Notably, the proliferation rate in resident stem cells in the bulb did not reduce and rather tended to increase after amputation of the tentacle (Revised Fig. 2D-2F), further supporting a scenario where resident stem cells do not migrate to blastema.Our new results together suggest that blastema contains highly proliferative cells, which are different from resident stem cells.Dedifferentiation has been observed in various organ regeneration in different species, and the initiation time of dedifferentiation after injury is distinct among different species and organs [1].
For example, during zebrafish fin regeneration, dedifferentiation begins at about 12 hours after injury [2] while during zebrafish heart regeneration, loss of sarcomeres in muscles and cell cycle re-entry, both of which indicate a hallmark of cardiomyocyte dedifferentiation, are observed 7 days after injury [3,4].In contrast, during the limb regeneration of adult newt, blastema begins to form 4-5 days after injury [5], and similarly, cell cycle re-entry in muscle is

Relative intensity
Intensity also observed 4 days after injury [6].Furthermore, the recent study on dedifferentiation in the cnidarian polyp Hydractinia has shown that it takes 3 days for cell cycle re-entry and 6 days for stem cell marker expression during head regeneration [7].Given that the time required for dedifferentiation is diversified among species and even among organs in the same species, the possibility that dedifferentiation occurs within 24 hours during tentacle regeneration in Cladonema jellyfish cannot be denied.
Comment 1.1.7:Irradiation that was used to eliminate resident i-cells wasn't effective enough as many i-cells remained (Figure S5C), making the results of these experiments inconclusive with respect to the source of i-cells in the blastema.
Response 1.1.7 We thank the reviewer for pointing out this issue.In the original Figure S5C, after 30 Gy X-ray irradiation, Nanos1 + stem cells still remained while the number of Nanos1 + cells significantly decreased.In this experimental condition, it is possible that the remaining resident stem cells contribute to blastema formation.To avoid this possibility, we have explored conditions that can remove most resident stem cells and found that 75 Gy X-ray irradiation causes near-total loss of Nanos1 + resident stem cells 3 days post-irradiation (dpi), which was confirmed by FISH and qPCR (Revised Fig. S7A-S7C and S7G).In addition to X-ray irradiation, we have further established a pharmacological treatment wherein Nanos1 + resident stem cells were almost completely eliminated after incubation with hydroxyurea (HU) 10 mM for 3 days or Mitomycin C 30 µM for 3 days (Revised Fig. S7D-S7G).In order to test whether blastema can appear after the removal of resident stem cells, we examined whether proliferative cells still accumulate to the wound site in these experimental conditions.At 24 hpa, EdU + proliferative cells accumulated to the regenerating tip, although the number of EdU + cells decreased slightly.
(Revised Fig. 4H-4J).These results indicate that blastema can form even in the absence of resident stem cells in the bulb and that most of the blastema cells are derived from non-resident stem cell lineage.We have revised the following sentence to explain the elimination of resident stem cells in the text: Firstly, as we have now repeatedly stated, we did not take the result of DiI labeling experiment as evidence of dedifferentiation but rather used it as a corroborating result to demonstrate that the contribution of resident stem cells to blastema formation is minimal.However, as the reviewer points out, in the original Figure S4C, we did not have a clear result from which to infer whether DiI labeling could stain resident stem cells (i-cells) in the bulb.We have thus performed both EdU labeling and β-catenin antibody staining in the same sample to confirm the cell-type of DiI+ cells in the bulb.We found that more than 75% of DiI labeled cells in the bulb are either EdU + cells or cells with i-cells morphology, as revealed by β-catenin cytoplasmic signal (Revised Fig. S6A-S6C).These results indicate that at least some portion of resident icells are labeled by DiI injection into the bulb.
To further confirm the origin of blastema cells, we injected DiI into either the bulb or the center of the tentacle to chase these DiI + cell populations while monitoring proliferating cells in blastema by EdU labeling (Revised Fig. S6D, S6E).We found that DiI + cells did not merge with EdU + cells in blastema when injected into the bulb (Revised Fig. S6Ei and S6Eii), whereas DiI + cells that were labeled in the center of the tentacle merged with EdU + cells in blastema (Revised Fig. S6Eiii and S6Eiv).(Note that DiI + cells in the center of the tentacle merged with EdU + cells in more than 80% of the tentacles observed, n=10/12 {Revised Fig.

S6D-S6E}
).These results suggest that blastema cells are not derived from resident stem cells in the bulb, but rather from the cells located close to the injury site.In fact, when DiI was injected into the bulb, DiI + cells did not migrate to the blastema area of the regenerating tentacle, but rather DiI + cells migrated to the tip of the regenerating tentacle including the nematocyte cluster (Revised Fig. S6G), supporting the idea that resident stem cells in the bulb differentiate into nematocytes, not regenerating epithelial cells.In contrast, when injected into the central tentacle area, DiI + cells were initially distributed near the blastema region and then distributed to the regenerating apical side (Revised Figure S6I), suggesting that blastema cells preferentially differentiate into epithelial cells.We have added the following sentences to the text to explain Response 1.1.9 As articulated above, we do not expect the different natures of blastema cells and resident stem cells to support the dedifferentiation scenario and rather have considered their differences to be evidence in support of distinct features of these stem-like populations.We confirmed that resident stem cells in the bulb differentiate into both epithelial cells and nematocytes, suggesting that they behave as multipotent stem cells, as in other hydrozoans (Revised Fig. 5A Considering the data in the revised manuscript, especially the additional evidence obtained during revision, it is clear that resident stem cells in the bulb do not significantly contribute to blastema formation and that proliferative cells in blastema behave in a repair-specific manner and exhibit a nature distinct from resident stem cells.Our main conclusion is that two stem-like cell populations contribute to tentacle regeneration with different roles, and thus we respectfully disagree with the suggestion to tone down the statements including the title.

Comment 1.2:
The resolution of most Figures in the reviewer PDF is poor.In Figure 4B, for example, it isn't possible to assess co-labeling by EdU/BrdU.Would it be possible to provide in the supplement some high-res, single confocal slices?If I got it right, 12.6% of the blastema cells were double positive.How many cells were double positive in the tentacle bud?Was it a similar percentage?
Response 1.2 The poor resolution of figures is probably due to the size limitation of the uploaded files, and these issues will be solved when submitting the final version that allows high resolution.
As requested, for Figure 4B, we have added a magnified image of single confocal slices to allow the evaluation of EdU and BrdU co-staining (Revised Fig. 4C).We have also quantified the number of EdU + & BrdU + (double positive) cells in the bulb region to clearly convey the results of the image analysis.In blastema, BrdU + repair-specific proliferative cells did not often merge with EdU + cells, which were labeled as resident stem cells (average 13.6%), whereas in the bulb, many BrdU + cells merged with EdU (average 68.7%) (Revised Fig. 4C-4D).These results suggest that resident stem cells in the bulb do not actively migrate to the injury site during the 24 hours after amputation.This also supports our main conclusion that resident stem cells and repair-specific proliferating cells in blastema are of different cellular origin with different characteristics.

Comment 1.3: Usage of 'ectoderm' and 'endoderm' to denote adult cnidarian external and
internal epithelial tissues, respectively, is misleading.Ectoderm and endoderm are embryonic lineages that give rise to specific tissues in the adult.There is no evidence in the literature from any cnidarian showing that their external tissue derives exclusively from their embryonic ectoderm and/or that their gastric tissue derives exclusively from endoderm.Use 'epidermis' and 'gastrodermis', or some other term that does not refer to embryonic origin, instead.

Response 1.3
We appreciate the reviewer for this constructive comment.We have changed the terms 'ectoderm' and 'endoderm' to 'epidermis' and 'gastrodermis,' respectively, in the revised manuscript."Subsequently, tentacles began to elongate distally, and nematocyte clusters, the spherical structures stained with the mature nematocyte marker poly-γ-glutamate, formed at the tip of the regenerating tentacle at 48 hpa (Figs 1Diii, 1Fiii, and S1A)." Comment 1.5: Line 412-413."24 hpa after amputation": "after amputation" is redundant.

Response 1.5
As suggested, we removed "after amputation" from the manuscript as follows: (Revised Manuscript; Page 15, Line 515-516) "Together, these observations suggest that cell proliferation is accelerated near the injury site at 24 hpa." Comment 1.6: Studied cnidarians have two Piwi-like genes.Which one has been studied here?
Regarding Nanos genes, some are Nematoblast rather than i-cell markers.Is Cladonema Nanos1 a likely i-cell marker?

Response 1.6
In contrast to some studied cnidarians [11,12], there is only one Piwi gene in Cladonema pacificum.The previous report by Hou et al. performed phylogenetic analysis to show the Piwi gene in Cladonema pacificum is homologous to the Piwi1 gene in Hydra vulgaris (Hydra vulgaris Piwi AFQ20832.1)and Podocoryna carnea (Podocoryna carnea Piwi AAS01181.1)[13].We thus used the Cladonema Piwi as a Piwi1 homologue in this study.
Regarding Nanos, there are two Nanos genes in Cladonema, Nanos1 and Nanos2, and we have utilized Nanos1 as a stem cell marker and Nanos2 as a nematoblast marker for the following reasons.
There are four lines of evidence supporting that Cladonema Nanos1 is a stem cell, or i-cell marker.First, Nanos1 has been employed as a marker for stem cells in various hydrozoan models including Hydra vulgaris and Clytia hemisphaerica [14,21].Second, Nanos1 is also predicted to be a stem cell marker in Cladonema medusa because Nanos1 is expressed at the branching site of the tentacles, in which more than 80% of the cells exhibit a stem cell morphology as shown in the previous work by Hou et al. [13].Third, we have compared the

It only shows S-phase cells; some may be committed progeny rather than stem cells (see also comment 1C). If the authors wish to identify the nature of S-phase cells, they should show EdU incorporation in conjunction
with an i-cell marker like Piwi1/2; they do show it in Figure 3E (with Nanon1) where many if not most Nanos1+ cells are EdU negative.

Response 1.7
In Figure 2, we intended to show the distribution pattern of proliferative cells, not stem-like cells, and that is why we showed only S-phase cells by EdU pulse labeling.Our results indicate that highly proliferative cells accumulated at the injury site (Revised Fig. 2Aii) and that cell proliferation is indeed more active at the amputation site than the bulb where resident stem cells are present (Revised Fig. 2D-2F), which together formed the foundation for further addressing cellular types and the role of blastema during tentacle regeneration.The cell types in blastema and their stem-like characteristics are described in detail in the revised Figure 3 and Figure S4.

Comment 1.8: Line 628. Not all hydrozoan i-cells are pluripotent. Hydra i-cells are multipotent
(the authors mention this in line 634).

Response 1.8
Indeed, not all hydrozoan i-cells exhibit pluripotency.Consequently, in the revised manuscript, we have modified the term "pluripotent" to "pluripotent or multipotent" (please see page 22, line 781).

Comment 1.9: Line 635. Watanabe et al. is not in the ref list.
Response 1.9 Thank you for noticing the reference omission.We have added the 'Watanabe et al.' to the reference list.

Response 1.10
We have replaced the term "homologous" with "similar" in line 723 of the original manuscript, which corresponds to line 892 in the revised manuscript.

Response 2.1
We thank the reviewer #2 for acknowledging the impact and quality of our work.
Regarding the novelty of our findings, we have shown that most repair-specific proliferative cells (RSPCs) in blastema during the Cladonema tentacle regeneration are not derived from resident stem cells while exhibiting i-cell morphological characteristics (Revised Fig. 3, 4, and S5-S7).We have further found that RSPCs express the stem cell markers Nanos1, Piwi, and Vasa1 with heterogeneous expression patterns among these cells, suggesting that RSPCs are heterogeneous stem-like cell population in terms of gene expression and likely their cellular characteristics (Revised Fig. 3Eiii, 3Eiv, 3F and S4C-S4E).Most importantly, we have demonstrated that the majority of RSPCs differentiate into epithelial cells in the newly regenerated tentacle whereas some RSPCs can still give rise to neurons (Revised Fig. S10), suggesting that RSPCs are multipotent, or at least bipotent, stem cells that preferentially generate epithelial cells.These results stand in stark contrast to the previous reports that describe pluripotent i-cells migrating to the injury site [9] or stem-like epithelial cells directly contributing to whole-body regeneration [22,23].
As suggested by the reviewers, studies using polyps like Hydractinia and Hydra have shown the existence of proliferating epithelial cells.During head regeneration of Hydractinia, cell proliferation occurs at the injury site.Although small numbers of epithelial cells can proliferate, more than 90% of proliferative cells are i-cells that migrate from the body column [9], which is totally different from Cladonema tentacle regeneration where RSPCs contribute to blastema.During head regeneration of Hydra after mid-gastric amputation, apoptosis occurs at the injury site, and the following cell proliferation is observed in the area immediately below the amputation site [24].In this case, most of these proliferating cells are indeed i-cells, and epithelial cells are rare.Intriguingly, when i-cell lineages (i-cells, neurons, nematocytes) are removed from Hydra, epithelial cells transiently proliferate at 18-30 hpa, and head regeneration seems to occur normally [22,23].It is noteworthy that the Hydra epithelial lineages are selfrenewing proliferative cells while their morphology and characteristics are distinct from i-cells [25], suggesting that epithelial cells in Hydra are not terminally differentiated cells, but rather stem cells, which may be a special feature of Hydra that is not conserved in other cnidarians.In contrast to the reviewer's understanding, regarding the sea anemone Nematostella and the jellyfish Clytia, existing literature lacks a definitive characterization of epithelial cell division after injury [16,[26][27][28][29], although these reports showed the requirement for cell proliferation and the involvement of stem cells during regeneration.
Taken together, our study is pioneering as it represents the first clear demonstration that RSPCs appear as a different population from resident stem cells and preferentially differentiate into epithelial cells during appendage regeneration.We have added the following sentence in the discussion: (Revised Manuscript; Page 23, Line 810-813) "While previous studies using other cnidarians have described active cell proliferation upon injuries, these proliferating cells are pluri/multipotent stem cells migrating to the injury [20,21,27,62], not lineage-restricted stem/progenitor cells like RSPCs."

Response to Reviewer 3
In

Response 3.1
In the original manuscript, the EdU images of 24-72 hpa were shown after 1 h incubation with EdU 20 µM, which was a different condition from the other experiments (e.g.revised Figure S5A).We thus replaced these images with the results of 1 h incubation with EdU 150µM (Revised Fig. 2A).In this condition, strong EdU signals were observed near the injury site at 24 hpa while relatively weak but recognizable EdU signals were detected in the bulb region of both intact and 24 hpa samples (please see Fig. 2A(i) and (ii)).
To clarify each pulse chase experiment using EdU and/or BrdU, we have added experimental schematics to the figures in the revised manuscript (Revised Fig. 4 S8A) although the ratio of EdU positive cells was similar to Revised Figure 5i wherein EdU 24 h incorporation was performed together with BrdU (Fig. R2).These results suggest that there is no significant difference of EdU incorporation across experiments.
Regarding Nowa FISH experiments, there was an error in the sequence that was used to design the FISH probe, so we have removed the corresponding data from the revised manuscript.

Comment 3.3.1:
Except for the qPCR in FigureS5A, all the quantifications in this paper are derived from microscopy images.While this is not a problem in and of itself, it does mean that the representative images chosen to accompany graphs need to be convincing.Unfortunately, there is repeated inconsistency throughout the paper between quantifications and the representative images.

Response 3.3.1
Due to the size limitation of the uploaded files, the quality of the figures might not provide sufficient quality, and some figures did not clearly show whether the signals were merged or not.To address the image issue, we have added enlarged images particularly for the quantified area as well as the size of the quantified area to figure legends (e.g.Revised Fig. 3E and 4C).In addition, to avoid inconsistency between quantifications and representative images, we have replaced some images that were less convincing (e.g.Revised Fig. 3E, 4C, 5B and 6B).For clarity, we have added EdU/BrdU magnified images of single confocal slices (Revised Fig. 4Ci, 4Cii) and further quantified the ratio of EdU + to BrdU + cells in both bulb and blastema (Revised Fig. 4D).The quantification showed that the EdU + /BrdU + cell ratio is 13.60±2.18% in blastema, although it is 68.66±4.82% in the bulb (Revised Fig. 4D).This result suggests that the proliferating cells in the bulb region at 24 hpa are still resident stem cells (resident homeostatic stem cells, RHSCs), but most blastema cells (repair-specific proliferative cells, RSPCs) are derived from a different lineage.We have changed the following sentence in the To clarify the image issue, we prepared single channel images of EdU for Figure S4E in the original manuscript (Revised Fig. S5C).Based on these images, we counted the number of EdU + cells in the whole tentacle at 24 hpa (Revised Fig. S5Ciii, S5D) as well as EdU + cells in intact tentacle (0h and 24 h no-amputation; Revised Fig. S5Ci, S5Cii, S5D).The size of the area where we counted EdU + cells in intact tentacle was similar to that of the regenerating tentacles at 24 hpa (Revised Fig. S5E), suggesting that following EdU incorporation, the number of EdU + cells increased similarly over 24 h in both no-amputation and regenerating tentacles.The quantification method and the average area of the quantified area were added to the figure Were these cells not counted?How were the % of Nanos1+ cells quantified?
Response 3.3.5 In the original manuscript, we did not count Nanos1 + cells in the middle area, and instead we counted Nanos1 + cells only in the tentacle bulb.To address the reviewer's concern, we have counted the number of Nanos1 + cells in the whole tentacle including the middle area (Revised Fig. S7A-S7C) and confirmed that after X-ray irradiation, Nanos1 + cells decreased in a dosedependent manner (Revised Fig. S7B-S7C).
Regarding the redistribution phenotype of Nanos1 + cells, indeed some Nanos1 + cells seemed to show redistribution to the middle area (Revised Fig. S7Bii: 30 Gy and iii: 50 Gy).
These surviving Nanos1 + cells may change localization as a response to irradiation, but at the current stage we have not pursued the cause as this topic is beyond this work's scope.More importantly, we have focused on the condition of 75 Gy X-ray irradiation for the subsequent experiments that address blastema formation after elimination of Nanos1 + cells (Revised Figure

Comment 1 . 1 . 4 :
Quiescent or slow cycling cells that re-enter the cell cycle post injury (the authors mention this possibility) would further reduce the numbers of double-labeled cells 24 hpa, making the dedifferentiation hypothesis even less likely.Response 1.1.4

(Comment 1 . 1 . 8 :
Revised Manuscript; Page 19, Line 650-667) "We first determined a condition that would eliminate resident stem cells after exposure to different doses of X-ray irradiation (30, 50, 75 Gy) by monitoring expression of the stem cell marker Nanos1.FISH staining of Nanos1 showed that Nanos1 + cells decreased in an X-ray dose dependent manor, and most Nanos1 + cells were removed at 75 Gy at 3 days post-irradiation (dpi) (S7A-S7C Fig).qPCR analysis further confirmed significant reduction of Nanos1 mRNA levels at 3 dpi (S7G Fig).We thus amputated the tentacle at 3 dpi and investigated the blastema formation by EdU pulse labeling (Fig4H).At 24 hpa, blastema successfully formed regardless of X-ray irradiation, although the rate of EdU + cells in the blastema decreased slightly (Fig 4Iand 4J).In order to support the conclusion derived from X-ray irradiation, we also performed pharmacological treatments using hydroxyurea (HU) or Mitomycin C, both of which are used to eliminate i-cells in other hydrozoan species[8][9][10].The results of FISH and qPCR confirmed that most Nanos1 + cells are removed by HU or Mitomycin C treatment for 3 days (Fig S7D-S7G).We then examined blastema formation after HU or Mitomycin C treatment and found that blastema formed at 24 hpa, consistent with the results of X-ray irradiation (Fig 4Iand 4J)."Next, the authors used DiI labeling of tentacle bulb tissue and find no labeled cells in the blastema 24 hpa.They take this as evidence for dedifferentiation.However, for this to be true they would have to show that the dye effectively labels i-cells in the tentacle bulb.icells are 'hidden' in interstitial spaces, and it is possible that only epithelial cells were labeled by the authors' method.Furthermore, nematocyte progenitors that do migrate to the blastema from the tentacle bulb according to the authors should have been visible if all cell types were labeled by DiI.Finally, FigureS4Cii shows only few DiI-labeled cells and all (or nearly all) of them are EdU negative, inconsistent with i-cell labeling.Response 1.1.8

Comment 1 . 1 . 9 :
the minimal contribution of resident stem cells based on DiI labeling experiments.(Revised Manuscript; Page 17-18, Line 599-609) "To confirm the cell-type of DiI + cells in the bulb, we first combined EdU staining or β-catenin antibody staining with DiI injection (S6A-S6C Fig) to show that more than 75% of EdU + cells or i-cells (β-catenin cytoplasm signal) were DiI + in the bulb (S6A-S6C Fig), indicating that the majority of resident i-cells are labeled by DiI injection.After a 24 h chase, DiI + cells were still localized near the bulb, not merged with EdU + cells in blastema (S6D and S6E Fig).In contrast, when DiI was injected into the central area near the amputation site, DiI + cells overlapped with EdU + cells, participating in blastema formation (S6D and S6E Fig).These results, taken togetherwith the EdU chase results, suggest that resident stem cells do not migrate to the blastema region."Lastly, the different nature of blastema cells as compared to tentacle bulb icells is interesting; however, it does not necessarily support the dedifferentiation hypothesis.An alternative interpretation of these results is that blastema cells are mainly committed epithelial progenitors (derived from tentacle bulb i-cells) while tentacle bulb i-cells are stem cells with broad developmental potential, like in other hydrozoans.

Comment 1 . 1 . 10 :
-5G).In contrast, repair-specific proliferative cells in blastema mainly contribute to newly generated tentacles by differentiating into epithelial cells (Revised Fig.6A-6C and S6H-S6I).Although the detailed molecular characteristics of repair-specific proliferative cells remains unspecified, the majority of them exhibit clear i-cell morphology (Revised Fig.3A-3B), suggesting that they are a part of hydrozoan i-cells based on current definition.Importantly, there is no clear marker for committed epithelial progenitors in cnidarians such as Cladonema, but instead these repair-specific proliferative cells heterogeneously express stem cell markers Nanos1, Piwi, and Vasa1 (Revised Fig.3E-3F and S4C-S4E).Intriguingly, we also found that repair-specific proliferative cells in blastema can differentiate into neurons in the regenerating tentacle (Revised Fig.S10C-S10D).We thus prefer to consider these proliferating blastema cells as lineage-restricted or bipotent stem cells based on i-cell morphological characteristics (Revised Fig.3A-3B) as well as differentiation potency (Revised Fig.6A-6C and S10C-S10D), unlike the multipotent resident stem cells in the bulb.The authors should revise their main conclusion by toning-down the corresponding statements on the source of blastema cells throughout the paper, including the title.It is of course possible that, in addition to migration, dedifferentiation also contributes to the blastema, but this remains a hypothesis, not supported by their current data.Response 1.1.10

Comment 1 . 4 :Response 1 . 4
Line 338.Nematocyte clusters are not visible on Figure 1D III.As we had not clearly defined nematocyte clusters, we have defined nematocyte clusters in tentacles with different resolutions in the revised Figure S1A.Both stereo and confocal microscope images show that the spherical structures are repeatedly formed on the tentacles (Fig S1Aii, S1Aiii), and they are stained with poly-γ-glutamate, which is a mature nematocyte marker, indicating that these spherical structures correspond to nematocyte clusters (Fig S1Aiii'-S1Aiii").Based on this criteria, Figure 1D (iii) shows a spherical bulge formed at the tip of the regenerating tentacle, indicating the newly formed nematocyte cluster.In order to clearly convey our intention, we have revised the manuscript as follows: (Revised Manuscript; Page 13, Line 431-434)

Comment 1 . 7 :
expression pattern of Nanos1 with other stem cell markers, Piwi and Vasa1, by FISH and found that Nanos1 was localized in the bulb and branching sites of tentacles along with Piwi and Vasa1 (Revised Fig. 3C, S3A-S3D, and S4A, S4B), suggesting that Nanos1 is one of the markers for stem cells.Fourth, double FISH showed that Nanos1 did not merge with Mcol1 or Nanos2, both of which are nematoblast markers (Revised Fig. S3E, see also the following paragraph).These findings strongly suggest that Nanos1 is an i-cell marker that can label part of stem cell populations.We have added the following sentence to the text in the revised manuscript.(Revised Manuscript; Page 16, Line 560-563) "Note that, while Cladonema possess two Nanos genes, Nanos1 and Nanos2, Nanos2 + cells co-express a nematoblast marker Mcol1, suggesting that Nanos2 is a nematoblast marker rather than a stem cell marker in Cladonema medusa (S3E and S3F Fig), similar to Hydractinia [47]."Line 421.No stem-like cells are shown in Figure 2.

Fig. R2 :
Fig. R2: Rate of EdU + cells in the bulb.EdU (150 µM/24 h) labeling from Figure 5B and Figure S8A.Quantification was performed on a single slice of each confocal image.The quantification area is 35 µm 2 .

Comment 3 . 3 . 2 :
The authors state that, at 24hpa, only 12.6% of EdU+ cells were also expressing BrdU.The images shown (Figure4Bi, i', and i'') do not support this claim.Better representative images, and perhaps an overlay of the BrdU/EdU channels at this time point are needed.

Comment 3 . 3 . 3 :Response 3 . 3 . 3 Comment 3 . 3 . 4 :
; Page 18, Line 622-624) "Importantly, only 13.6% of BrdU + RSPCs were EdU + in blastema, supporting the notion that the migration of RHSCs may not be sufficient to form blastema (Fig 4Bi, 4Cii, and 4D)."In Figure3, the Nanos1+/EdU+ cell images in E do not corroborate the quantifiations in F. Including a zoomed in image of the double positive cells would better support the claim, as the current images are impossible to interpret.To address the reviewer's concern, we have added enlarged images (Revised Fig.3Eii and 3Eiv) that corroborate the quantification of Nanos1 + /EdU + cells (please see the revised Fig.3E, 3F).This result shows that the majority of EdU + cells (69.7%) co-express Nanos1 in the bulb whereas fewer EdU + cells (30.3%) co-express Nanos1 in blastema, supporting our proposal that resident homeostatic stem cells and repair-specific proliferative cells are of different lineages.FigureS4Eshows a 2-fold increase in the number of EdU+ cells, but the images in panel D do not support these numbers.

Comment 3 . 3 . 5 :
legend as follows: (Revised Manuscript; Page 40, Line 1392-1396) "(D) The number of EdU + cells labeled before amputation.Counted area is the whole regenerating tentacle at 24 hpa.Detailed information is in (E).Intact: n = 6 (tentacles), 24 h (no amp): n = 6, 24 hpa: n = 6.(E) Area size used for quantification.Note that the area of quantification at 24 hpa is similar." Figure S5C claims that the % Nanos1+ cells decrease upon irradiation, but the image in panel B instead seems to show a redistribution towards the middle of the image.

Reviewer #2 Comment 2.1: Fujita
et al investigated the cellular mechanisms of tentacle regeneration in the hydrozoan medusa Cladonema pacificum.They demonstrate that after tentacle ablation, highly of epithelial cells at the site of injury were shown to happen in several cnidarians (notably Hydra, Hydractinia, Clytia and Nematostella), it is not so surprising that those proliferating cells are not only migrating cells but also local epithelial cells re-entering the cell cycle.It remains an interesting paper, providing further information on an additional cnidarian species.
this manuscript, Fujita et al. describe the process of blastema formation in the medusa The expression level of Mcol1 was extremely high, and indeed the FISH Mcol1 signal (Cy5) invaded the channel for EdU signal (Alexa Fluor 488).We thus adjusted the intensity of EdU to become darker, resulting in a different view than appears in other figures.In order to clarify the EdU signals, we have added the magnified figures and single slice images (Revised Fig.S8A).